The KGE motif in FVIIa is required for integrin β1 complex formation and proangiogenic cell signaling. (A) Location of the surface-exposed KGE motif in the crystal structure of FVIIa.68 (B) Amidolytic activity of FVIIa wt and E26A was measured by chromogenic substrate conversion (n = 4). P = .503, Student t test. (C) HaCaT cell-surface FXa generation with indicated concentrations of FVIIa E26A and FVIIa wt. (D) PAR2 receptor cleavage by FVIIa wt and E26A as well as trypsin in Chinese hamster ovary cells expressing FLAG-tagged PAR2 (n = 3). ***P < .001, one-way ANOVA and Dunnett’s test. (E) TS2/16 immunoprecipitation of Brij 35 lysates from HaCaT cells stimulated with 10 nM FVIIa wt or E26A for 30 minutes. Upper panel: representative western blots of TF in TS2/16 immunoprecipitates and CLs. Lower panel: densitometric quantification of TF western blots (n = 6). *P < .05, one-way ANOVA and Dunnett’s test. (F) Time course of ERK phosphorylation in HaCaT cells stimulated with 10 nM FVIIa wt or E26A. Upper panel: representative western blots of pERK and total ERK. Lower panel: densitometric quantification of pERK western blots normalized to total ERK of 3 independent experiments (n = 3). *P < .05, ***P < .001, Student t test. (G) Quantification of IL-8 mRNA in HaCaT cells stimulated for 90 minutes with 10 nM FVIIa wt and E26A, respectively (n = 8). ***P < .001, one-way ANOVA. (H) TF cell-surface expression of HaCaT cells stimulated with FVIIa wt and E26A determined by fluorescence-activated cell sorting (FACS) analysis (n = 7). **P < .01, one-way ANOVA. (I) Confocal imaging of HaCaT cells incubated with 10 nM FVIIa wt and E26A, respectively, in the presence of TS2/16-Alexa 647 conjugate (5 μg/mL). Cells were stained for early endosomes with αEEA1-Alexa488 (5 μg/mL) and nuclei (Hoechst, 1 μg/mL) after fixation. Scale bar = 10 μm. MFI, mean fluorescence intensity; n.s. not significant.
